A survey of enabling technologies in synthetic biology Kahl and Endy

Kahl and Endy Journal of Biological Engineering 2013, 7:13 http://www.jbioleng.org/content/7/1/13 Kahl and Endy Journal of Biological Engineering 2013, 7:13 http://www.jbioleng.org/content/7/1/13

RESEARCH Open Access A survey of enabling technologies in synthetic biology Linda J Kahl* and Drew Endy

Abstract Background: Realizing constructive applications of synthetic biology requires continued development of enabling technologies as well as policies and practices to ensure these technologies remain accessible for research. Broadly defined, enabling technologies for synthetic biology include any reagent or method that, alone or in combination with associated technologies, provides the means to generate any new research tool or application. Because applications of synthetic biology likely will embody multiple patented inventions, it will be important to create structures for managing intellectual property rights that best promote continued innovation. Monitoring the enabling technologies of synthetic biology will facilitate the systematic investigation of property rights coupled to these technologies and help shape policies and practices that impact the use, regulation, patenting, and licensing of these technologies. Results: We conducted a survey among a self-identifying community of practitioners engaged in synthetic biology research to obtain their opinions and experiences with technologies that support the engineering of biological systems. Technologies widely used and considered enabling by survey participants included public and private registries of biological parts, standard methods for physical assembly of DNA constructs, genomic databases, software tools for search, alignment, analysis, and editing of DNA sequences, and commercial services for DNA synthesis and sequencing. Standards and methods supporting measurement, functional composition, and data exchange were less widely used though still considered enabling by a subset of survey participants. Conclusions: The set of enabling technologies compiled from this survey provide insight into the many and varied technologies that support innovation in synthetic biology. Many of these technologies are widely accessible for use, either by virtue of being in the public domain or through legal tools such as non-exclusive licensing. Access to some patent protected technologies is less clear and use of these technologies may be subject to restrictions imposed by material transfer agreements or other contract terms. We expect the technologies considered enabling for synthetic biology to change as the field advances. By monitoring the enabling technologies of synthetic biology and addressing the policies and practices that impact their development and use, our hope is that the field will be better able to realize its full potential. Keywords: Synthetic biology, Biological engineering, Enabling technologies, Survey, Intellectual property rights, Licensing, Regulation

* Correspondence: [email protected] Bioengineering Department, Stanford University, Y2E2 Room 269C, 473 Via Ortega, Stanford, CA 94305-4201, USA

© 2013 Kahl and Endy; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 2 of 18 http://www.jbioleng.org/content/7/1/13

Background coupled to those technologies. Investors and funders of Synthetic biology is an emerging, interdisciplinary field synthetic biology research also may find this information that aims to make the design, construction, and optimi- useful in guiding funding decisions and establishing pol- zation of biological systems easier and more reliable. icies for the patenting and licensing of enabling technolo- Advances in synthetic biology will deepen our under- gies. Information gained from monitoring the enabling standing of how biological systems work, and should technologies of synthetic biology also may be useful in enable faster and cheaper development of useful medi- identifying technology trends, and could help government cines, chemicals and materials, new means for informa- agencies and non-governmental organizations in crafting tion processing and data storage, and sources of food policy frameworks to address the safety and security con- and energy that could help promote human health and cerns raised by synthetic biology research. preserve the environment. Although the field of syn- thetic biology is relatively young, there have already been Results promising advances in engineering microorganisms to Demographic data produce important drugs [1,2], exploring strategies for During the period the survey was active, from August biofuel production [3,4], and designing and DNA-based 31, 2012 to January 30, 2013 a total of 160 responses information storage [5,6] and genetically-encoded com- were received. Six responses were excluded because they munications and processing systems [7,8]. did not contain answers to any of the substantive ques- As is true for many emerging fields of research, realiz- tions on technology use. Seventeen responses were from ing the full potential for constructive applications of participants who answered “no” to Survey Question 11 synthetic biology will require not only the continued that asked survey participants to indicate whether they development of enabling technologies but also the im- considered themselves to be a synthetic biologist or to plementation of policies and practices to ensure that be engaged in basic or applied synthetic biology research these technologies remain accessible to those working in or development. Responses from these 17 survey partici- basic and applied research. The enabling technologies pants were analyzed separately, and the remaining 137 for synthetic biology can be defined, broadly, as any re- responses were used for most analyses. agent or method that, alone or in combination with as- Responses originated from the United States (121 re- sociated technologies, provides the means to generate sponses, 88%) and ten other countries (16 responses, any new research tool or application in synthetic biology. 12%). The distribution of responses from outside the Because the field of synthetic biology spans a wide range United States was Australia (1), Canada (1), Germany (2), of disciplines – from engineering and biology to math- Israel (1), Italy (1), Japan (3), Mexico (1), Sweden (1), and ematics and computer science – the technologies con- the United Kingdom (5). Responses were received from sidered “enabling” by synthetic biology researchers may researchers working exclusively in a non-commercial be expected to cover a broad range, depending on the organization (n = 91, 66%), exclusively in a commercial focus and nature of the research. Monitoring the enab- organization (n = 39, 28%) and in both commercial and ling technologies of synthetic biology is an important non-commercial organizations (n = 7, 5%). Among survey step towards understanding the needs, abilities and ac- respondents working exclusively in a non-commercial complishments of this diverse research community. organization, most indicated that they worked in a college Here, we conducted a survey among a self-identifying or university (n = 64), research institution (n = 9), govern- community of practitioners engaged in synthetic biology ment laboratory (n = 1), were affiliated with both a col- research to obtain their opinions and experiences with lege/university and research institution (n = 12) or were technologies that support the engineering of biological independent (n = 5). Among survey respondents working systems. The aim of this first study was to define a set of exclusively in a commercial organization, most were from enabling technologies for the field of synthetic biology, small companies of fewer than 50 employees (n = 21) and with a focus on technologies used in research laborator- the rest were from companies of more than 1000 em- ies in both academia and industry. Our goal was to ployees (n = 11), fewer than 1000 employees (n = 3), and gather information about the technologies considered fewer than 250 employees (n = 4). Of the 7 survey respon- enabling by practitioners in the field so that we and dents that worked in both commercial and non-commer- others might better evaluate the landscape of synthetic cial organizations, all worked in a small company of fewer biology and explore policies and practices that best pro- than 50 employees as well as a college/university or re- mote continued innovation. For example, it is likely that search institution. useful applications of synthetic biology will embody mul- tiple patented inventions and monitoring the enabling Experience with the iGEM competition technologies of synthetic biology will facilitate the sys- Survey Question 2 asked participants to provide infor- tematic investigation of the intellectual property rights mation about their experience as student or non-student Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 3 of 18 http://www.jbioleng.org/content/7/1/13

participants of the International Genetically Engineered The majority of synthetic biology researchers in academia Machines (iGEM) competition (http://igem.org), a syn- (n = 88) used biological parts from or contributed parts to thetic biology competition aimed at undergraduate stu- theiGEMRegistry(n=60,68%)andtheATCC(n=53, dents, high school students, and entrepreneurs. Of the 60%), and many used or contributed to Addgene (n = 42, 136 survey participants that responded to this question, 48%) (Figure 1A). Other publicly available registries that 112 (82%) indicated that they did not participate in the were widely used among academic researchers included the iGEM competition as a student, 21 (15%) indicated that CGSC (n = 18, 20%), the SynBERC Registry (n = 13, 15%), they previously participated in the iGEM competition as JBEI-ICE Public (n = 11, 12%) and EUROSCARF (n = 8, a student, and 3 (2%) indicated that they were a student 9%). Fewer researchers in academia reported use or contri- currently participating in the iGEM competition. bution of parts to the ARS/NRRL (n = 5), the BIOFAB In addition, 58 survey participants provided free-text responses describing their experience as non-student participants of the iGEM competition. Of these, 49 stated that they had advised or mentored iGEM teams or served as judges for the iGEM competition, 6 sup- ported or sponsored iGEM teams, and 3 stated that they had other experience with the iGEM competition, in- cluding participating in organizing the software division, assisting in evaluation of the iGEM program, and read- ing iGEM research reports Thirteen of the 58 survey participants who provided free-text responses were also former students of the iGEM competition, while 45 had no prior experience with iGEM as students.

Use of publicly available registries Survey Question 3 asked participants to indicate whe- ther they used publicly available registries to obtain nat- ural or engineered biological materials or information. This question allowed survey participants to select from a list of publicly available registries and to write in any additional registries of which they were aware. The listed registries included publicly available collections of infor- mation (including DNA sequences) or tangible materials that could be used for synthetic biology research, includ- ing plasmids encoding specific biological functions, DNA-binding proteins, microorganisms and cell lines (hereinafter referred to as biological parts). Specifically, the publicly available registries initially listed included the Registry of Standard Biological Parts supporting the iGEM competition (iGEM Registry), the American Type Culture Collection (ATCC), Addgene, the Coli Genetic Stock Center (CGSC), the Synthetic Biology Enginee- ring Resource Center (SynBERC) Registry, the Joint BioEnergy Institute Public Registry (JBEI-ICE Public), the European Saccharomyces cerevisiae Archive for Functional Analysis (EUROSCARF), the Agricultural Research Service NRRL collection (ARS/NRRL), the BIOFAB: International Open Facility Advancing Biotech- Figure 1 Publicly available registries of natural or engineered biological materials or information. (A) Percentage of synthetic nology (BIOFAB), the Dana-Farber/Harvard Cancer biology researchers in academia that use biological parts from, or Center (DF/HCC) PlasmID Repository, the DNASU contribute parts to, publicly available registries. (B) Percentage of Plasmid Repository (DNASU), the Belgian Coordinated synthetic biology researchers in industry that use biological parts Collections of Micro-organisms (BCCM), and the from, or contribute parts to, publicly available registries. (C) Impact Leibniz-Institut DSMZ - German Collection of Microor- of iGEM experience on use of the iGEM Registry by synthetic biology researchers in academia and industry. ganisms and Cell Cultures (DSMZ). Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 4 of 18 http://www.jbioleng.org/content/7/1/13

(n = 2), the DF/HCC (n = 2), the DNASU (n = 2), the others. Most synthetic biology researchers in academia DSMZ (n = 1), and the BCCM (n = 1). Additional regis- reported that the laboratory or organization in which tries identified by academic researchers included the they worked maintained its own registry of biological CyanoBase-Kazusa Genome Resources (http://www.ncbi. parts (55/88, 62%) (Figure 2). Of these, the vast majority nlm.nih.gov/pmc/articles/PMC2808859) (n = 1), and the of academic researchers made these materials available various yeast collections distributed by Invitrogen (n = 1). to others outside their own laboratory (53/55, 96%). A Synthetic biology researchers in industry (n = 39) significantly greater proportion of academic researchers reported different usage rates for publicly available regis- sent parts directly to others (40/53, 75%) as compared to tries (Figure 1B). The majority of industry researchers those that distributed parts through a publicly available used or contributed parts to the ATCC (n = 26, 67%), registry (15/53, 28%) (p < 0.00001). and many used or contributed parts to the iGEM Regis- Similarly, most synthetic biology researchers in indus- try (n = 18, 46%). Other publicly available registries used try reported that the laboratory or organization in which by industry researchers included Addgene (n = 12, 31%), they worked maintained its own registry of biological the CGSC (n = 9, 23%), the SynBERC Registry (n = 4, parts (21/39, 54%). However, fewer than half of industry 10%), the JBEI-ICE Public (n = 3, 8%) and the ARS/ researchers made these parts available to others (9/21, NRRL (n = 4, 10%). Fewer industry researchers reported 43%). A significantly greater proportion of industry use or contribution of parts to the EUROSCARF (n = 2), researchers also sent materials directly to others (6/9, the BCCM (n = 1), and the DSMZ (n = 2). Additional 67%) as opposed to distributing parts through a publicly registries identified by industry researchers included the available registry (1/9, 11%) (p = 0.05). Keio/ASKA collection of E. coli strains (http://www. A comparison of the use and distribution rates be- shigen.nig.ac.jp/ecoli/strain/top/top.jsp) (n = 1) and the tween academic and industry researchers revealed no pZ series expression vectors developed by Lutz and statistically significant difference in the likelihood of Bujard [9] (http://www.expressys.com) (n = 1). maintaining a private registry of biological parts (p = Among the publicly available registries included in this 0.43). However, researchers in academia were signifi- survey, only the iGEM Registry showed a statistically sig- cantly more likely to make parts available to others than nificant difference in usage rates between researchers in researchers in industry (p < 0.000001). academia and industry (p = 0.03). This difference was explored further by examining the impact of prior iGEM experience on use of the iGEM Registry (Figure 1C). Favorite or most useful biological parts Among synthetic biology researchers in academia, those A total of 43 participants responded to Survey Question having experience with the iGEM competition either as 5, which was an open question asking respondents to list a student or non-student participant (e.g., advisors, their favorite or most useful biological parts (Table 1). judges, sponsors, etc.) were significantly more likely to Biological parts that survey participants identified in contribute parts to or use parts from the iGEM Registry more general terms included the Anderson promoter li- as compared to academic researchers without iGEM brary (n = 4), in-house promoters (n = 2), three-color experience (93% and 46%, respectively, p < 0.00001). Similarly, industry researchers having experience with the iGEM competition were significantly more likely to use or contribute parts to the iGEM Registry than indus- try researchers lacking iGEM experience (65% and 26%, respectively, p = 0.02). Academic researchers having iGEM experience also were significantly more likely than industry researchers with iGEM experience to use or contribute to the iGEM Registry (93% and 65%, respect- ively, p < 0.01). No significant difference was observed in use of the iGEM Registry between academic and in- dustry researchers without iGEM experience (46% and 26%, p = 0.17). Figure 2 Private registries of natural or engineered biological Use of private registries materials or information. Percentage of synthetic biology Survey Question 4 asked participants to indicate whe- researchers in academia and industry that maintain a private registry ther the laboratories or organizations in which they of biological parts, make these materials available to others, send worked maintained a private registry of biological parts, materials to others directly, and send materials via a publicly available registry. and whether and how these parts were made available to Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 5 of 18 http://www.jbioleng.org/content/7/1/13

Table 1 Favorite or most useful biological parts from publicly available registries ID Category N Description Addgene 25712:pAKTaq Plasmid 1 Bacterial expression vector encoding the DNA polymerase from Thermus aquaticus Coli Genetic Stock Center CGSC #12119 Chassis 1 E. coli strain BW27783 bearing 9 known mutations iGEM Registry BBa_J23100 series Regulatory 8 BBa_J23100 through BBa_J23119 is a family of constitutive promoter parts that can be used to tune the expression level of constitutively expressed parts BBa_B0034 RBS 4 RBS based on Elowitz & Liebler repressilator BBa_B0015 Terminator 1 Double terminator including BBa_B0010 and BBa_B0012 BBa_C0062 Coding 1 luxR repressor/activator BBa_E2050 Coding 1 derivative of mRFP1, yeast-optimized

BBa_F2620 Signaling 1 A signaling device whereby the input is 3OC6HSL and the output is PoPS from a LuxR-regulated operator BBa_I15010 Coding 1 Chimeric Cph1 light receptor/EnvZ protein BBa_I744210 Generator 1 TetR regulated LuxN-Tsr Chimeric Receptor B BBa_J04450 Reporter 1 RFP coding device BBa_J15001 RBS 1 strong synthetic E. coli RBS with SacI site BBa_J153000 Plasmid Backbone 1 broad-host-range shuttle vector pPMQAK1 that provides ampicillin and kanamycin/neomycin resistance BBa_J176005 Protein Domain 1 Codon optimized mCherry red fluorescent protein BBa_J176006 Coding 1 Mammalian venus fluorescent protein BBa_J176022 Protein Domain 1 Human codon-optimized AmCyan1 from pAmCyan1-C1 BBa_J33207 Reporter 1 lac promoter and lacZ BBa_J61009 Plasmid 1 pAC-LuxGFP that places GFP under the wildtype Vibrio lux device BBa_J64032 Device 1 pCASP SPI-1 Secretion Circuit BBa_J85226 Composite 1 Kanamycin resistance (KanR)_off version of J85224 BBa_J176027 Regulatory 1 Constitutive cytomegalovirus promoter BBa_J176122 Plasmid Backbone 1 pcDNA3.1 plus puromycin resistance BBa_K566002 Regulatory 1 Biphasic switch BBa_P1010 Generator 1 ccdB cell death gene BBa_R0040 Regulatory 1 TetR repressible promoter scaffold for monitoring gene expression (n = 1), dose- methods. This question allowed survey participants to dependent promoters (n = 1), BIOFAB promoters (n = select from a list of physical assembly methods and to 1), fluorescent proteins (n = 9), colored proteins made at write in any additional assembly methods that they used. DNA2.0 (n = 1), all the working reporters in the iGEM Of the 134 survey participants that answered this ques- Registry (n = 1), BioBrick vectors (n = 2), pRS vector tion, most indicated that they currently use or previously series (n = 1), pZ vectors (n = 1), vectors (n = 1), high have used the Gibson assembly method (48% current, copy plasmid backbones from the iGEM Registry (n = 22% past) and de novo DNA synthesis (50% current, 18% 1), bicistronic design parts (n = 1), quorum sensing parts past) (Figure 3). The original BioBrick standard (18% (n = 1), terminator variants (n = 1), and TetR (n = 2). current, 31% past) and Gateway cloning (15% current, 26% past) were selected by a significant number of sur- Use of physical assembly standards and methods vey participants, although most indicated past use of Survey Question 6 asked participants to indicate their these specific methods. Survey participants indicated current and past use of physical assembly standards and lower overall usage rates for other physical assembly Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 6 of 18 http://www.jbioleng.org/content/7/1/13

Table 2 Additional physical assembly methods identified by survey participants Physical Assembly Method N Commercial/Proprietary Proprietary method, not specified 3 GeneArt Seamless Assembly 2 GeneArt High Order Assembly 1 Clontech In-Fusion HD cloning kit 1 Ginkgo assembly method 1 Invitrogen TOPO cloning 1 Non-Commercial Conventional PCR [10,11]10 Yeast in vivo recombinational cloning [12]5 Home-brew method, not specified 4 Figure 3 Physical assembly standards and methods. Current and Restriction-site Associated DNA (RAD) assembly [13]2 past use of physical assembly standards and methods by synthetic Anderson 2 antibiotic (2ab) assembly [14]1 biology researchers. Inverse PCR [15]1 Splicing by Overlap Extension (SOE) [16]1 methods, including the BglBrick standard (16% current, 16% past), Sequence and Ligase Independent Cloning In development (SLIC) (14% current, 21% past), GoldenGate (16% A new enzymatic, scarless synthesis and assembly technology 1 current, 10% past), and others. Extensions of BioBytes assembly standard [17]1 In addition to the physical assembly methods listed in Question 6, survey participants providing free-text re- Additional measurement tools identified by survey partic- sponses to this question (n = 33) identified additional as- ipants included fluorescence reporter protein measurement sembly methods that they used in the course of (n = 4), Miller assay (n = 2), beta-galactosidase assay synthetic biology research (Table 2). (n = 2), cell auto-fluorescence (n = 1), dual-luciferase re- porter measurement (n = 1), comprehensive metabolite measurement (n = 1), comprehensive proteome (n = 1), Use of measurement, functional composition and data specific mRNA or protein measurements (n = 1), RNAseq exchange standards and tools (n = 1), and qPCR (n = 1). Additional data exchange tools A total of 120 and 127 survey participants responded to included custom laboratory management information sys- Survey Questions 7 and 8, respectively, which asked re- tems (n = 2), JERM (n = 1), RightField (n = 1), Systems spondents to indicate their current or past use of meas- Biology Markup Language (SMBL) (n = 1), GoogleDocs urement tools, functional composition standards, and (n = 1), and the GenBank file format (n = 1). data exchange tools. These questions allowed survey participants to select from a list of tools and to write in any additional measurement, functional composition and data exchange tools that they used. Usage rates for measurement standards, functional composition stan- dards, and data exchange standards were relatively low (Figure 4). Specifically, the number of survey respon- dents reporting current and past use were: Relative Pro- moter Unit (RPU) (7% current, 14% past), Polymerase Per Second (PoPS) (2% current, 14% past), Relative Mammalian Promoter Unit (RMPU) (0 current, 2% past), Expression Operating Unit (EOU) (4% current, 3% past), Synthetic Biology Open Language (SBOL) (18% current, 10% past), SBOL visual (SBOLv) (16% current, Figure 4 Standards and methods for measurement, functional 3% past), JBEI-ICE repository platform (8% current, 2% composition, and data exchange. Current and past use of past), electronic datasheets (7% current, 7% past), and standards and methods for measurement, functional composition, and data exchange by synthetic biology researchers. visual datasheets (4% current, 4% past). Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 7 of 18 http://www.jbioleng.org/content/7/1/13

Use of additional tools, reagents and methods reaction (PCR), green fluorescent protein (GFP), and Survey Question 9 asked participants to indicate their non-GFP reporter molecules, while fewer researchers current and past use of additional tools, reagents, and used newer techniques such as directed evolution (e.g., methods. This question allowed survey participants to MAGE) (22% current, 10% past). select from a list and to write in any other tool reagent In addition to the tools, reagents and methods that or method that they considered enabling for the field of were listed, survey participants providing free text re- synthetic biology. Of the survey participants that sponses (n = 23) identified additional technologies that responded to this question, the vast majority indicated they considered enabling for synthetic biology (Table 3). that they used GenBank as their preferred genomic data- base (84% current, 12% past) and employed tools for Use of software tools search (84% current, 15% past), alignment (78% current, A total of 133 survey participants responded to Survey 16% past), and analysis (68% current, 13% past) of DNA Question 10, which asked participants to indicate their sequences (Figure 5). Most survey participants used current and past use of software tools. This question commercial DNA synthesis services for short oligos allowed survey participants to select software tools that (86% current, 11% past) and gene-size fragments (71% were listed and to write in any additional software tools current, 12% past), while fewer researchers used in- they used. The highest rates of current use were reported house DNA synthesis for short oligos (14% current, 15% for ApE (41% current, 24% past), Primer 3 (33% current, past) and gene-size fragments (26% current, 44% past). 24% past), Mfold (34% current, 21% past), and the Ribo- The vast majority of survey participants indicated that some Binding Site (RBS) Calculator (28% current, 28% they used established culture techniques, as well as other past) (Figure 6). Vector NTI (17% current, 51% past), established technologies such as the polymerase chain GeneDesigner (22% current, 27% past), and Mathematica (11% current, 35% past) were selected by a significant number of survey participants, although most indicated past use of these software tools. Lower but increasing-over -time usage rates were reported for the j5 DNA Assembly (16% current, 9% past), Genome Compiler (11% current, 6% past), and GenoCAD (10% current, 6% past) software tools. In addition to the software tools that were listed, survey participants providing free text responses (n = 36) identified additional software tools used in the course of their synthetic biology research (Table 4).

Technology choices and self-identification as a synthetic biologist Survey Question 11 asked participants whether they considered themselves to be a synthetic biologist or to be engaged in basic or applied synthetic biology research or development. Because this question was introduced on Day 6 of the survey, not all participants were able to respond to this question. A total of 58 survey partici- pants had submitted responses prior to Day 6 and because all of these participants were students or post- doctoral fellows in the Endy or Smolke labs or re- searchers affiliated with SynBERC they were considered to be synthetic biology researchers for the purposes of the survey. Of the 96 survey participants that submitted responses on Day 6 and later, 79 answered “yes” and 17 answered “no” to this question. Responses from the 79 participants that affirmatively self-identified as synthetic Figure 5 Additional tools, reagents and methods. Current and biologists and the 58 participants that responded prior past use of genomic databases, sequence tools, DNA synthesis tools, to Day 6 were grouped together for most analyses. Re- DNA sequencing tools, culture techniques, and other tools, reagents, sponses from the 17 participants that did not self- and methods by synthetic biology researchers. identify as synthetic biologists were analyzed separately. Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 8 of 18 http://www.jbioleng.org/content/7/1/13

Table 3 Additional technologies considered enabling for synthetic biology by survey participants Tool, Reagent or Method Description (URL or reference) N BioCyc a collection of 1962 pathway and genome databases (http://biocyc.org)1 Bioinformatics the application of computational techniques to analyze the information associated with biomolecules on 1 a large-scale (http://www.ncbi.nlm.nih.gov/About/primer/bioinformatics.html) CAGE: Conjugative Assembly a technology that permits the hierarchical consolidation of modified genomic regions [18]1 Genome Engineering High Throughput Computing the ability to run many copies of software at the same time across many different computers, 1 reviewed in [19] in vitro screens tests for biological activity such as metal binding screens, electron uptake, and other enzymatic activity 2 IonTorrent an approach to DNA sequencing that enables a direct connection between chemical and digital 1 information and aims to place DNA sequencing within the reach of any laboratory or clinic [20] EcoCyc a database for Escherichia coli K-12 MG1655 (http://ecocyc.org)1 Flow Cytometry a technology that uses the principles of light scattering, light excitation, and emission of fluorochrome 3 molecules to generate specific multi-parameter data from particles and cells in the size range of 0.5 μmto 40 μm diameter (http://crl.berkeley.edu/flow_cytometry_basic.html) KEGG: Kyoto Encyclopedia of Genes a database resource for understanding high-level functions and utilities of the biological system, such as 1 and Genomes the cell, the organism and the ecosystem, from genomic and molecular-level information (http://www. genome.jp/kegg) Mass spectrometry a technology for targeted protein quantification, reviewed in [21]2 MetaCyc a database of nonredundant, experimentally elucidated metabolic pathways (http://metacyc.org)1 Molecular biology technologies, includes methods and reagents for creating competent cells, nucleic acid transfer, digestion, primer 9 generally extension, ligation, assembly of DNA molecules, etc. OptForce an algorithm that identifies all possible metabolic interventions that lead to the overproduction of a 1 biochemical of interest [22] PDB: Protein DataBank an information portal to biological macromolecular structures (http://www.rcsb.org/pdb/home/home.do)1 Protein purification technologies methods for purifying a protein of interest efficiently, reviewed in [23]1 Recombineering an in vivo method of genetic engineering applicable to chromosomal and episomal replicons 1 in E. coli [24] Robotic automation use of robots for repetitive laboratory tasks such as pick and place, liquid and solid additions, heating, 2 cooling, mixing, shaking, etc. Single cell microscopy a technology that enables visualization of gene expression with exquisite spatial and temporal sensitivity, 1 reviewed in [25] Standards, needed includes standards for calibrating and sharing data from plate readers, standards for test, measurement 3 and characterization, standards for documentation and sharing of biological modules, for example see Arkin, 2008 [26] and Endy, 2005 [27] SOLiD a next generation sequencing technology that allows identification of hundreds of millions of short RNAs 1 in a sample in a single run [28] Transcription Activator-Like (TAL) a technology that allows proteins to be designed to specifically target and bind to a desired sequence 1 effector technology of DNA [29] UniProt: Universal Protein Resource a collaboration between the European Bioinformatics Institute (EBI), the SIB Swiss Institute of 1 Bioinformatics and the Protein Information Resource (PIR) that aims to provide a comprehensive resource for protein sequence and annotation data (http://www.uniprot.org) Yeast in vivo recombination methods for assembling large DNA constructs in the yeast Saccharomyces cerevisiae, for example see 1 Gibson et al., 2008 [30] and Jaschke et al., 2012 [31]

Of the 17 participants that did not self-identify as syn- employees, 2 in a company of fewer than 50 employees, thetic biologists, 12 were from the US, 2 were from the and 1 in a company of fewer than 250 employees. UK, 1 was from Austria, 1 was from Norway, and 1 was Twelve of the 17 participants had no experience with from Portugal. Eleven participants worked exclusively in the iGEM competition, 1 had been a sponsor for iGEM non-commercial organizations – 5 in a college or uni- teams, and 1 collaborated with an iGEM team as a DIY versity, 2 in a research institution, 1 in both a college/ biologist. university and research institution, and 3 were independ- Among the 17 participants who did not self-identify as ent. The remaining 6 worked exclusively in commercial synthetic biologists, two indicated that they used parts organizations – 3 in a company with more than 1,000 from the iGEM Registry (one was a former iGEM Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 9 of 18 http://www.jbioleng.org/content/7/1/13

engineering, and more. Our survey tapped the experi- ences and perspectives of this diverse research commu- nity in order to glean initial insights into the technologies that are considered enabling for the field by its practitioners. The results of the survey offer a first snapshot view of the technologies previously and now in use by synthetic biology researchers, and give a sense of the many and varied technologies that support work in synthetic biology. One of our objectives in conducting this survey was to establish a set of technologies considered enabling for the field of synthetic biology, so that we and others might systematically investigate the intellectual property rights coupled to these technologies. For example, one overall consideration regarding enabling technologies and property rights is whether or not these technologies are accessible for use – either by virtue of being in the public domain or through legal tools such as non- exclusive licensing – to researchers in academic, govern- ment, and commercial organizations. The extent to which innovation in synthetic biology, and biotechnol- ogy more generally, may be impeded by broad founda- tional patents that cannot be licensed or patent thickets remains unclear [32-36]. Identifying the technologies to which wide, unrestricted access is needed to promote continued innovation in synthetic biology is an import- ant step towards understanding the impact of patenting Figure 6 Software tools. Current and past use of software tools by synthetic biology researchers. and licensing practices on access to the enabling tech- nologies underlying this field. Consistent with the postulate that past scientific student, the other had no iGEM experience). In addition, achievements lay the foundation for future innovation, some participants used parts from other publicly avail- the results of the survey showed that many of the tech- able registries, including the ATCC (n = 5), Addgene nologies that enable research in synthetic biology are (n = 3), the ARS/NRRL (n = 2), the BIOFAB (n = 1), and well established and in the public domain. For many of the CGSC (n = 1). With regards to private registries, 4 these earlier technologies patent protection was either participants (3 from academia, 2 from industry) indi- not sought or, even if patent protected, sufficient time cated that the laboratories or organizations in which has lapsed for the technologies to enter the public do- they worked maintained a private registry of biological main. For example, the vast majority of survey respon- parts. Of these, 3 made parts available to others (all from dents reported use of bacterial cell culture technologies academia) – 3 by sending parts directly, and 1 by also such as LB medium or glycerol freezing (Figure 5), yet distributing parts through a publicly available registry. these technologies were published in the scientific litera- None of these 17 participants reported current use of ture as early as the 1950’s [37-39] and are squarely in standards and methods for measurement, functional the public domain. Similarly, the vast majority of survey composition, or data exchange, although past use of the respondents reported use of PCR technology, yet ele- RPU (n = 1), electronic datasheets (n = 1), and visual ments of PCR technology have entered the public datasheets (n = 1) was noted. Both current and past use domain or will do so shortly. Specifically, foundational was reported for other technologies covered in this sur- patents covering amplification methods (e.g., US vey, including physical assembly methods, software tools, 4,683,195 and EP 0 200 362 B), thermal cycling instru- and other tools, reagents, and methods (Table 5). ments (e.g., US 5,038,852 and EP 0 395 736 B), and thermostable DNA polymerases (e.g., US 4,889,818 and Discussion EP 0 258 017 B) have now expired. Although patents The emerging field of synthetic biology has captured the continue to be filed on improvements to PCR technolo- interest and energy of researchers from a variety of gies, many subsequent patents such as those covering disciplines – biology, chemistry, computer science, thermostable polymerases with enhanced activities (e.g. Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 10 of 18 http://www.jbioleng.org/content/7/1/13

Table 4 Additional software tools used for synthetic biology research by survey participants Software tool Description (URL) N ABySS: Assembly By Short a de novo, parallel, paired-end sequence assembler that is designed for short reads 1 Sequences (http://www.bcgsc.ca/platform/bioinfo/software/abyss) AlignDNA a pairwise DNA alignment tool (http://www.geneinfinity.org/sms/sms_aligndna.html)1 BLAST: Basic Local Alignment a program that finds regions of local similarity between biological sequences (http://blast.ncbi.nlm.nih.gov)7 Search Tool CentroidFold a program that predicts an RNA secondary structure from an RNA sequence (http://www.ncrna.org/centroidfold)1 CLC Genomics Workbench a comprehensive and user-friendly analysis package for analyzing, comparing, and visualizing next generation 5 sequencing data (http://www.clcbio.com/products/clc-genomics-workbench) CodonCode Aligner a program for sequence assembly, contig editing, and mutation detection (http://www.codoncode.com/aligner)1 Cytoscape an open source software platform for visualizing and integrating complex networks (http://www.cytoscape.org)1 FastPCR a program for PCR primer design (http://en.bio-soft.net/pcr/FastPCR.html)1 Gene Construction Kit a program for plasmid mapping (http://www.textco.com/gene-construction-kit.php)1 Geneious a program for handling and managing bioinformatics data (http://www.geneious.com)7 Gibthon Ligation Calculator a software tool for calculating reactant concentrations for DNA ligation (http://django.gibthon.org/tools/ligcalc)1 JWS online a tool for simulation of kinetic models from a curated model database (http://jjj.biochem.sun.ac.za)1 Lasergene-DNAStar comprehensive software for DNA and protein sequence analysis, contig assembly and sequence project 7 management (http://www.dnastar.com) Mascot a search engine which uses mass spectrometry data to identify proteins from primary sequence databases 1 (http://www.matrixscience.com/search_intro.html) Mauve a system for efficiently constructing multiple genome alignments in the presence of large-scale evolutionary 1 events such as rearrangement and inversion (http://gel.ahabs.wisc.edu/mauve) Merlin a M.A.G.E. optimization tool developed by the Cross-disciplinary Integration of Design Automation Research 1 group at Boston University (http://cidar1.bu.edu:8080) OligoAnalyzer software for comprehensive oligonucleotide analysis (http://www.idtdna.com/analyzer/Applications/ 1 OligoAnalyzer) ORF Finder: Open Reading a graphical analysis tool which finds all open reading frames of a selectable minimum size in a user’s sequence 1 Frame Finder or in a sequence already in the database (http://www.ncbi.nlm.nih.gov/projects/gorf) PaR-PaR software that allows researchers to use liquid-handling robots effectively (http://prpr.jbei.org)1 Pigeon Synthetic Biology Open Language picture generator (http://cidar1.bu.edu:5801/pigeon1.php)1 PlasMapper software that automatically generates and annotates plasmid maps using only the plasmid DNA sequence as 1 input (http://wishart.biology.ualberta.ca/PlasMapper) PyMOL a user-sponsored molecular visualization system for rendering and animating 3D molecular structures on an 1 open-source foundation (http://pymol.org) RNAstructure a complete package for RNA and DNA secondary structure prediction and analysis (http://rna.urmc.rochester. 1 edu/RNAstructure.html) Serial Cloner freeware with an intuitive interface that assists in DNA cloning, sequence analysis and visualization 3 (http://serialbasics.free.fr/Serial_Cloner.html) Sequencher DNA sequencing software (http://genecodes.com/sequencher-features)1 SSC: Stochastic Simulation a tool for creating exact stochastic simulations of biochemical reaction networks (http://web.mit.edu/irc/ssc)1 Compiler SWISS-PDB viewer (aka an application that provides a user friendly interface allowing analysis of several proteins at the same time 1 DeepView) (http://spdbv.vital-it.ch) Synbiota a platform of collaborative services to design, store, post, organize, access, or share information 2 (https://mozillalabs.com/en-US/synbiota) Velvet a set of algorithms to manipulate de Bruijn graphs for genomic sequence assembly (http://www.ebi.ac.uk/ 1 ~zerbino/velvet)

US 5,436,149 and US 5,556,772) have either expired or protected or have patent applications pending, yet are are due to expire within the next year. accessible through non-exclusive licensing. For example, Other more recently developed technologies used by the majority of survey respondents reported current or synthetic biology researchers are currently patent past use of the Gibson assembly method [40] for in vitro Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 11 of 18 http://www.jbioleng.org/content/7/1/13

Table 5 Technologies used by survey participants that did not self-identify as synthetic biologists (n = 17) Tool, reagent or method Current use Past use Tool, reagent or method Current use Past use Physical assembly methods Genomic database Gateway recombinatorial cloning 4 1 GenBank 10 3 de novo DNA synthesis 3 2 E!EnsemblGenomes 7 3 Gibson assembly 3 1 MicrobesOnline 3 1 Conventional restriction site-based cloning 2 0 European Nucleotide Archive 2 2 CPEC 1 2 DNA Databank of Japan 2 0 SLIC 1 1 Sequence tools PIPE 1 1 Search (e.g., BLAST) 12 3 USER 1 1 Alignment (e.g., ClustalW2) 10 1 InFusion cloning 1 0 Analysis (e.g., OligoCalc) 7 2 RAD assembly 1 0 Software tools Transfer PCR 1 0 ApE 6 1 Yeast in vivo cloning 1 0 Rosetta 4 1 BioBrick assembly standard 0 1 Vector NTI 3 5 Measurement, functional composition, data exchange Primer3 3 3 RPU 0 1 Mathematica 2 5 Electronic datasheets 0 1 Mfold 1 2 Visual datasheets 0 1 Gene Designer 1 2 DNA synthesis Blast 1 1 Commercial, short oligos 9 4 J5 DNA Assembly 1 1 Commercial, gene size (>500bp) 4 3 Vector Editor 1 1 In-house, gene-size(>500 bp) 3 2 GenoCAD 1 1 In-house, short oligos 1 4 Cell Designer 1 0 DNA sequencing ClothoCAD 1 0 Commercial 8 3 iBioSim 1 0 In-house 2 4 Gene Design 1 0 Culture technique ProtoBiocompiler 1 0 LB broth or agar 10 3 SimBiology 1 0 37°C incubator 10 3 SnapGene 1 0 Antibiotic selection 9 4 TinkerCell 1 0 Glycerol freezing 8 3 GenomeCompiler 0 1 30°C incubator 7 3 GEntle 0 1 Colorimetric medium 4 2 GLAMM 0 1 Other tools, reagents and methods COPASI 0 1 PCR 9 5 DeviceEditor 0 1 GFP reporters 7 4 Lasegene-DNA Star 0 1 Non-GFP reporters 6 4 RBS Calculator 0 1 Directed evolution 3 2 physical assembly of DNA constructs (Figure 3). Several is expected to extend through at least 2026, access to granted patents and pending patent applications are the Gibson assembly method has been made available relevant to this method, including US Patents 7,723,077 through a non-exclusive licensing agreement between and 7,776,532, US Applications 2010/0184187 and 2010/ Synthetic Genomics, Inc. and New England BioLabs, 0311126, and International Application PCT/US2006/ Inc. [41]. As such, components may be purchased from 031214. Although the exclusive period for these patents New England BioLabs, Inc., albeit with significant Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 12 of 18 http://www.jbioleng.org/content/7/1/13

restrictions that limit use to “internal research purposes well as many of the others listed in the survey, provide for the sole benefit of the purchaser only” [42]. Those researchers with tangible materials (e.g., cultures, plas- desiring additional rights to the Gibson assembly mids, and other reagents) as well as information relevant method, for example to manufacture commercial prod- to the material (e.g., source, nucleic acid sequence, per- ucts, must contact Synthetic Genomics, Inc. directly. formance specifications). From a technical perspective, Still other technologies that survey respondents consider public registries are useful only to the degree that the enabling for the field of synthetic biology are heavily pa- biological materials contained within are reliable and ac- tent protected and the ability of researchers to access these curately described. Towards that end, several publicly technologies through licensing is less clear. For example, available registries have undertaken steps to curate the the majority of survey respondents indicated current or parts received and to verify nucleic acid sequence infor- past use of GFP or non-GFP reporter molecules (Figure 5). mation. From an intellectual property perspective, access Fluorescent proteins are commonly used as genetically to materials from public registries is limited not only by encoded reporter molecules that enable researchers to considerations of patent protection, but also by the observe the activity of particular genetic elements and bio- terms of material transfer agreements or other contracts molecules inside live cells or tissues. One of the founda- that may govern the transfer of tangible materials. Regis- tional patents covering uses of GFP (US 5,491,084) is due tries of biological parts such as the SynBERC registry, to expire in September 2013. However, the exclusivity pe- the JBEI-ICE Public, and the BIOFAB currently provide riods for other foundational patents on GFP and its uses information only. To the extent that the genetically- are expected to continue for a number of years (e.g., US encoded materials described in these registries may be 5,741,668, US 6,146,826, EP 0 759 170 B1). Furthermore, readily synthesized from the sequence information pro- there are literally hundreds, if not thousands, of issued vided, use of these materials is limited primarily by con- patents covering variants of GFP and their uses. For ex- siderations of patent protection. Although the SynBERC ample, a search of CAMBIA’s Patent Lens (http://www. and JBEI-ICE Public registries currently indicate whether patentlens.net) for the term “green fluorescent protein” in the material is “encumbered” or “not encumbered,” no the same claim [i.e., Expert Search of (green near/2 fluor- other information is provided to assist researchers wish- escent) and (fluorescent near/2 protein) and (green near/2 ing to use these materials with identifying relevant pat- protein) in claims] yielded 770 granted US patents and ents. As for private registries, many survey respondents 256 granted European patents. Similar searches for yellow indicated that they shared materials with others and that fluorescent protein, red fluorescent protein, and blue they distributed materials by direct transfer as well as fluorescent protein yielded over 400 patents granted in the through publicly available registries. The types of mate- US and Europe. The large number of patents covering var- rials maintained in private registries and the terms for iants of GFP and their uses presents a considerable chal- their transfer were not queried in this survey, though lenge to synthetic biology researchers who wish to use considerations of patent protection and possibly add- fluorescent reporters in creating standards for characteriz- itional contract terms would be relevant to use of mate- ing biological parts and devices, such as the Relative Pro- rials from private registries as well. moter Unit (discussed below). Some relief to navigating Not all of the technologies queried in this survey were this thicket of patents may be available through negotiat- used by the majority of survey participants. Unlike the ing a license agreement with Life Technologies or GE high usage rates reported for physical assembly stan- Healthcare. For example, the ATCC has recently an- dards and methods (Figure 3, Table 2), relatively few sur- nounced that they have secured a license agreement that vey participants indicated current or past use of enables them to distribute GFP-containing biological ma- standards and methods for measurement, functional terials to non-commercial and government researchers composition, and data exchange (Figure 4). One possibil- [43]. However, for-profit customers must have a separate ity that could account for such relatively low usage rates license with Life Technologies or GE Healthcare to obtain is that some types of standards and methods have been and use these materials. introduced only recently. For example, the EOU has In addition to the observations noted above, the survey been presented at meetings as early as 2010 [44], but an results indicate several trends for the use of technologies initial formal description and example applications of by synthetic biology researchers. The majority of survey the EOU have only recently been published [45,46]. An- respondents reported that they used biological parts other possibility that could account for the relatively low from or contributed parts to publicly available registries usage rate is that some types of standards and methods (Figure 1) as well as private registries maintained within require tools that are not readily licensed by industry. individual laboratories (Figure 2). Among the most For example, the RPU requires measurement of fluores- widely used publicly available registries were the iGEM cent reporter molecules [47] and it may be necessary to Registry, the ATCC, and Addgene. These registries, as work through the patent thicket surrounding uses of Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 13 of 18 http://www.jbioleng.org/content/7/1/13

GFP before widespread adoption of such standards is provide more balanced insight into the technologies possible. Finally, it may also be that some technologies considered enabling for synthetic biology. Second, al- identified in this survey are useful to only a subset of though in-person interviews were conducted to identify survey respondents. The synthetic biology research com- relevant technologies and create questions for the sur- munity is comprised of diverse group of individuals with vey, the majority of responses were obtained through an varied interests and goals. As such, the technologies that online format. Without an interactive format, such as are considered vitally important for the work of some re- in-person or telephone interviews, to clarify any ambigu- searchers may not be used at all by others. ities in the wording of survey questions, respondents To the extent that one or more patented technologies may have misunderstood some of the questions posed. could eventually become widely adopted as a standard in Third, the survey questions focused on the technologies the field, it may be advisable for the synthetic biology re- actually used by the synthetic biology research commu- search community to consider creating more formal nity and did not explore the potential reasons underlying organizational structures and to articulate policies and why certain technologies were not used. Additional best practices for the disclosure and licensing of pat- questions that directly query whether intellectual prop- ented technologies. Although standardization in syn- erty rights covering certain technologies represented a thetic biology is still at a relatively young stage [48,49], selection barrier against the use of those technologies there are indications that the patent landscape is becom- would also be informative. Finally, the results of this ing quite complex [50]. To mitigate potential difficulties survey reflect the experiences of synthetic biology re- in the development and implementation of standards searchers at only one point in time. Re-administration of using patented technologies, the field of synthetic biol- surveys such as the one developed here could provide a ogy could benefit from the lessons learned by the more complete view of the technologies that are consid- information and communications technologies (ICT) in- ered enabling for synthetic biology as the field develops dustry, where a multitude of patented technologies have over time. been incorporated into standards [51]. There, the cre- Beyond facilitating the systematic investigation of ation of standards development organizations with for- property rights, monitoring the enabling technologies of mal policies requiring fair, reasonable and non- synthetic biology could also help inform governmental discriminatory (F/RAND) licensing terms have helped to and non-governmental organizations in crafting policy alleviate some of the problems the ICT industry has frameworks to address the safety and security concerns faced in incorporating patented technologies into stan- raised by innovation in this field. Access to concrete data dards [52]. Because the creation of a standards develop- on the technologies used by those working in basic and ment organization is not a trivial undertaking and could applied synthetic biology research can be vital for mak- potentially raise antitrust concerns, it will be important ing changes to existing policies as well as for creating to work with counsel and abide by the recommendations new options for governance. For example, advances in of governmental agencies [53,54]. DNA synthesis technology and the resulting commercial Several limitations should be taken into account when availability of larger synthetic DNA constructs [56] have interpreting the results from this survey. First, this sur- led to a shift from research conducted with recombinant vey sampled only a fraction of the global synthetic biol- DNA to research conducted with synthetic nucleic acid ogy research community and included responses from molecules. This shift in the technologies used for re- 137 individuals, mostly from universities or research in- search, in turn, has prompted the U.S. Department of stitutes within the United States. It is difficult to esti- Health and Human Services to issue amended guidelines mate the number of individuals conducting research in for research involving recombinant or synthetic nucleic synthetic biology, though one study identified nearly acid molecules [57] and to develop recommendations 3,000 authors of scientific publications who were work- for a framework for synthetic nucleic acid screening ing on or writing about synthetic biology [55]. Given the [58]. Similarly, monitoring the enabling technologies of relatively small sample size, the survey results may be synthetic biology could help alert policy makers and subject to sampling bias (i.e., the demographics of the stakeholders to advances in technology that may exert a survey respondents may not accurately reflect the demo- comparable impact on innovation and research practices graphics of the synthetic biology research community) in this field. as well as potential reporting biases (i.e., the voluntary reporting of this survey necessarily excludes those who Conclusion chose not to volunteer responses). A larger sampling of The survey results presented here provide insight into the the synthetic biology research community, with greater enabling technologies of synthetic biology. As innovation representation of researchers outside of the United in this field continues to advance we expect that the re- States as well as researchers working in industry, might agents, methods, and tools considered enabling for Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 14 of 18 http://www.jbioleng.org/content/7/1/13

synthetic biology will change. The policies and practices of ments based on responses received by Days 6 and 19. government, funding, and community organizations that On Day 19 and at various times thereafter, a link to the impact the regulation, patenting, and licensing of these survey was forwarded to additional researchers working technologies are also subject to change. Because research in the field of synthetic biology by the BioBricks Founda- in synthetic biology is conducted across multiple institu- tion (BBF), a public benefit organization that represents tions in many countries, it will be important to adopt pol- the public interest in the field of synthetic biology icies and practices that promote cross-institutional and (http://biobricks.org), the ERASynBio, a program for the transnational exchange of ideas, data, and technology. development and coordination of synthetic biology in Moreover, because it is likely that useful applications the European Research Area (http://www.erasynbio.eu), of synthetic biology will embody multiple patented in- the iGEM Foundation, a public benefit organization that ventions, it will be important to create structures for organizes the iGEM competition (http://igem.org), the managing intellectual property rights that will pro- organizers of SynBioBeta, an industry conference for mote access to the technologies used to comprise and synthetic biology startup companies (http://synbiobeta. create commercially available products. By monitoring com), and individuals working in community biolabs. the enabling technologies of synthetic biology and ad- The survey was available via an interactive website vancing policies and best practices for the patenting, (http://www.surveymonkey.com) or as a Word docu- licensing and regulation of these technologies, our ment directly from the authors, and the responses hope is that the field will be better able to reach its reported were collected from August 31, 2012 through full potential to promote human health and preserve January 30, 2013. Instructions provided at the beginning the environment. of the survey encouraged respondents to answer the questions based on their own experience and perspec- Methods tive. A PDF file of the survey questions is available as Survey design, distribution and analysis Additional file 1. We designed a web-based survey soliciting responses on Questionnaire data were exported into Microsoft Excel technologies that could be considered enabling by prac- for analysis and only valid responses were evaluated (i.e., titioners engaged in synthetic biology research. For pur- only responses to the specific questions were included in poses of the survey, enabling technologies were defined each analysis). Analyses that involved comparison of as tools, reagents, and methods that, alone or in com- researchers in academia and industry included only re- bination with associated technologies, provide the means sponses from survey participants that worked exclusively to generate any new research tool or application in syn- in a non-commercial or commercial setting. Survey par- thetic biology. Technologies included in the survey were ticipants were considered to be working in a non- compiled through review of the scientific literature and commercial setting if they indicated that they worked in personal interviews with synthetic biology researchers a college or university, research institute, government la- from both academia and industry. Researchers working boratory, or were independent (e.g., citizen scientist, in the field of synthetic biology were located by means amateur biologist, etc.). Survey participants were consid- of personal references, professional networking, and re- ered to be working in a commercial setting if they indi- ferrals from synthetic biology organizations. For this first cated that they worked in a for-profit company of any survey, we focused on technologies used in research size. Statistical significance was evaluated using Fisher’s 1laboratories for the engineering of biological systems. exact test over binary contingency tables [60]. Given its seminal importance in promoting a sense of community [59], we also examined the role of the iGEM Registries of natural or engineered biological materials or competition in fostering the adoption of certain tech- information nologies by synthetic biology researchers. Technologies Eleven publicly available registries were listed in Ques- associated with safety and security were not within the tion 3 throughout the duration of the survey: the iGEM scope of this survey, nor were other potentially enabling Registry (http://partsregistry.org), the JBEI-ICE Public resources such as professional societies or technology (https://public-registry.jbei.org), the SynBERC Registry roadmaps. (https://registry.synberc.org), Addgene (http://www. We first piloted the survey by sending the question- addgene.org), the DNASU Plasmid Repository (http:// naire to researchers working in the laboratories of Drs. dnasu.asu.edu/DNASU/Home.jsp), the DF/HCC Plas- Christina Smolke and Drew Endy in the Bioengineering mID Repository (http://plasmid.med.harvard.edu/PLAS- Department at Stanford University, and made adjust- MID), the ATCC (http://www.atcc.org), the CGSC ments based on initial responses. We then sent the sur- (http://cgsc.biology.yale.edu), the EUROSCARF (http:// vey to members of the Synthetic Biology Engineering web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html), Resource Center (SynBERC) and made further adjust- the Félix d’Hérelle Reference Center for Bacterial Viruses Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 15 of 18 http://www.jbioleng.org/content/7/1/13

(http://www.phage.ulaval.ca/no_cache/en/accueil), and the ena), GenBank (http://www.ncbi.nlm.nih.gov/genbank), ARS/NRRL culture collection (http://nrrl.ncaur.usda.gov). e!EnsemblGenomes (http://www.ensemblgenomes.org), This group of publicly available registries includes both and MicrobesOnline (http://www.microbesonline.org). registries that distribute tangible materials (e.g., cell lines, Tools for searching, alignment, and analysis of DNA plasmids, DNA binding proteins) and registries that serve sequences such as BLAST [84], ClustalW2 [85], and solely as repositories of information (e.g., DNA se- OligoCalc [86], respectively, as well as commercial quences, plasmid construction, performance specifica- and in-house methods for DNA synthesis [74] and tions). Two additional publicly available registries were DNA sequencing [87] were also included. Long- added to the list for Question 3 after the collection of established cell culture technologies included anti- 85 responses, based on free text responses – the BCCM biotic selection, temperature selection, lysogeny broth (http://bccm.belspo.be/index.php) and the DSMZ (a.k.a., Luria-Bertani medium or LB medium) [37,88], (http://www.dsmz.de). colorimetric media [89], and glycerol freezing of bac- terial strains [38,39]. More recently established tools Physical assembly standards and methods such as PCR [10,11], fluorescent reporter molecules Sixteen physical assembly standards and methods were [90], and directed evolution [91] were also included. listed in Question 6 throughout the duration of the survey: the original BioBrick assembly standard (BBF RFC 10) [61], the BglBrick assembly standard (BBF RFC 21) [62], the Software tools BioFusion standard (BBF RFC 23) [63], Freiberg standard Thirty software tools, many of which have been recently (BBF RFC 25) [64], the AarI cloning standard (BBF RFC reviewed[92],werelistedinQuestion10throughoutthe 28) [65], the BioBytes assembly standard (BBF RFC 47) duration of the survey: ApE (http://biologylabs.utah.edu/ [17], Circular Polymerase Extension Cloning (CPEC) [66], jorgensen/wayned/ape), BioJADE (http://web.mit.edu/jago DNA assembler [67], Gateway recombinatorial cloning ler/www/biojade), BioNetCAD (http://www.sysdiag.cnrs.fr/ [68], Gibson assembly [40], GoldenBraid assembly [69], BioNetCAD), Cell Designer (http://celldesigner.org), GoldenGate shuffling [70], Modular Cloning (MoClo) [71], ClothoCAD (http://www.clothocad.org), COPASI (http:// Seamless Ligation Cloning Extract (SLICE) [72], Sequence www.copasi.org), DeviceEditor (replaced by AutoBioCAD; and Ligase Independent Cloning (SLIC) [73], and de novo http://j5.jbei.org/index.php/Main_Page), Eugene (http:// DNA synthesis [74]. Two additional physical assembly eugenecad.org), GEC (http://research.microsoft.com/en-us/ methods were added to the list in Question 6 after the col- projects/gec), Gene Designer (https://www.dna20.com/ lection of 52 responses based on free text responses – genedesigner2), GeneDesign (http://www.genedesign.org), Polymerase Incomplete Primer Extension (PIPE) [75] and GenoCAD (http://www.genocad.org), Genetdes (http://jara Uracil Specific Excision Reagent (USER) [76]. millolab.issb.genopole.fr/display/sbsite/Download), GLAMM (http://glamm.lbl.gov), iBioSim (http://www.async.ece.utah. Tools for measurement, functional composition and data edu/iBioSim), j5 DNA Assembly Design Automation Soft- exchange ware (http://j5.jbei.org/index.php/Main_Page), Mathematica Tools supporting functional composition of genetic (http://www.wolfram.com/mathematica), Mfold (http://mfo objects, measurement of intracellular molecular ld.rna.albany.edu/?q=mfold), OptCircuit (http://maranas.che. activities, and data exchange were listed in Survey psu.edu/research_circuits.htm), Primer3 (http://simgene. Questions 7 and 8. Measurement tools included Poly- com/Primer3), ProMoT (http://www.mpi-magdeburg.mpg. merase Per Second (PoPS) [77], relative promoter unit de/projects/promot), ProtoBiocompiler (http://proto.bbn. (RPU) [47], relative mammalian promoter unit com/Proto/Proto.html), RBS Calculator (https://salis.psu. (RMPU) [78], functional composition tools included edu/software), Rosetta (http://www.rosettacommons.org), the expression operating unit (EOU) [45], and data RoVerGeNe (http://iasi.bu.edu/~batt/rovergene/rovergene. exchange tools included Synthetic Biology Open Lan- htm), SimBiology - MATLAB (http://www.mathworks.com/ guage (SBOL) [79], SBOL Visual (SBOLv) [80,81], and products/simbiology), SynBioSS (http://www.synbioss.org), visual or electronic data sheets for biological parts TinkerCell (http://www.tinkercell.com), VectorEditor (http:// and devices [82,83]. j5.jbei.org/index.php/Main_Page), and Vector NTI (http:// www.invitrogen.com/site/us/en/home/Products-and-Servi Additional tools, methods and reagents ces/Applications/Cloning/vector-nti-software.html). Three Additional tools, reagents, and methods that survey additional software tools were added to the list for participants could consider enabling were listed in Question 10 after the collection of 51 responses based Survey Question 9. Genomic databases included the on free text responses – GenomeCompiler (http://www. DNA Databank of Japan (http://www.ddbj.nig.ac.jp), genomecompiler.com), GENtle (http://gentle.magnusman European Nucleotide Archive (http://www.ebi.ac.uk/ ske.de), and SnapGene (http://www.snapgene.com). Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 16 of 18 http://www.jbioleng.org/content/7/1/13

Additional file 7. Ortiz ME, Endy D: Engineered cell-cell communication via DNA messaging. J Biol Eng 2012, 6:16. 8. Bonnet J, Yin P, Ortiz ME, Subsoontorn P, Endy D: Amplifying genetic logic Additional file 1: PDF file of the survey questions. gates. Science 2013, 340:599–603. 9. Lutz R, Bujard H: Independent and tight regulation of transcriptional Abbreviations units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 – ARS/NRRL: Agricultural research service NRRL culture collection; regulatory elements. Nucleic Acids Res 1997, 25:1203 1210. ATCC: American type culture collection; BBF: BioBricks foundation; 10. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, et al: Primer-directed BCCM: Belgian coordinated collections of micro-organisms; BIOFAB: BIOFAB enzymatic amplification of DNA with a thermostable DNA polymerase. – international open facility advancing biotechnology; CGSC: Coli genetic stock Science 1988, 239:487 491. center; DF/HCC: Dana-farber/harvard cancer center; DSMZ: Leibniz-Institut 11. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, et al: Enzymatic DSMZ - German collection of microorganisms and cell cultures; amplification of beta-globin genomic sequences and restriction site – EOU: Expression operating unit; EUROSCARF: European Saccharomyces analysis for diagnosis of sickle cell anemia. Science 1985, 230:1350 1354. cerevisiae archive for functional analysis; GFP: Green fluorescent protein; 12. Oldenburg KR, Vo KT, Michaelis S, Paddon C: Recombination-mediated iGEM: International genetically engineered machines; JBEI-ICE Public: Joint PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res 1997, – BioEnergy institute public registry; LB medium: Luria-bertani medium; 25:451 452. MoClo: Modular cloning; PIPE: Polymerase incomplete primer extension; 13. Etter PD, Preston JL, Bassham S, Cresko WA, Johnson EA: Local de novo PoPS: Polymerase per second; RBS: Ribosome binding site; RMPU: Relative assembly of RAD paired-end contigs using short sequencing reads. PLoS mammalian promoter unit; RPU: Relative promoter unit; SLIC: Sequence and One 2011, 6:e18561. ligase independent cloning; SLICE: Seamless ligation cloning extract; 14. Leguia M, Brophy J, Densmore D, Anderson JC: Automated assembly of SynBERC: Synthetic biology engineering resource center; SBOL: Synthetic standard biological parts. Methods Enzymol 2011, 498:363–397. biology open language; SBOLv: SBOL visual; SMBL: Systems biology markup 15. Ochman H, Gerber AS, Hartl DL: Genetic applications of an inverse language; USER: Uracil specific excision reagent. polymerase chain reaction. Genetics 1988, 120:621–623. 16. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR: Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap Competing interests extension. Gene 1989, 77:61–68. DE is a co-founder and director of a commercial DNA assembly company 17. Lloyd D, Robinson K, Garside E, Fedor J, Ridgway D, et al: BBF RFC 47: BioBytes (Gen9, Inc.). No other competing interests have been declared by the Assembly Standard. 2009. http://dspace.mit.edu/handle/1721.1/49518. authors. 18. Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, et al: Precise manipulation of chromosomes in vivo enables genome-wide codon ’ Authors contributions replacement. Science 2011, 333:348–353. LJK and DE conceived the study. LJK designed the survey, analyzed the 19. Morgan M, Grimshaw A: High-throughput computing in the sciences. responses, and wrote the manuscript. DE provided critical feedback and Methods Enzymol 2009, 467:197–227. suggestions during the development and analysis of the survey. Both 20. Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, et al: An integrated authors reviewed and approved the final manuscript. semiconductor device enabling non-optical genome sequencing. Nature 2011, 475:348–352. Acknowledgements 21. Pan S, Aebersold R, Chen R, Rush J, Goodlett DR, et al: Mass spectrometry We thank all of the participants that responded to the survey questions, based targeted protein quantification: methods and applications. members of the Endy and Smolke labs for contributing to the development J Proteome Res 2009, 8:787–797. of the survey, and Kevin Costa at the SynBERC, Holly Million at the BBF, Andy 22. Ranganathan S, Suthers PF, Maranas CD: OptForce: an optimization Boyce at the BBSRC and the ERASynBio, Meagan Lizarazo and Randy procedure for identifying all genetic manipulations leading to targeted Rettberg at the iGEM Foundation, John Cumbers and Lisa Comeau at the overproductions. PLoS Comput Biol 2010, 6:e1000744. SynBioBeta conference, Mackenzie Cowell at DIYbio, and Antony Evans at 23. Walls D, McGrath R, Loughran ST: A digest of protein purification. Glowing Plant for their efforts in promoting the survey. We also thank Paul Methods Mol Biol 2011, 681:3–23. Jaschke and Monica Ortiz for sharing their technological expertise, Nicolas 24. Sharan SK, Thomason LC, Kuznetsov SG, Court DL: Recombineering: a Kahl for insight on motivating survey participants, Markus Sommer and Eddie homologous recombination-based method of genetic engineering. Moler for advice on statistical analysis, and Megan Palmer for helpful Nat Protoc 2009, 4:206–223. discussions throughout the project. Funding for this work was provided by 25. Larson DR: What do expression dynamics tell us about the mechanism of the NSF-sponsored Synthetic Biological Engineering Research Center transcription? Curr Opin Genet Dev 2011, 21:591–599. (SynBERC) and Stanford University. 26. Arkin A: Setting the standard in synthetic biology. Nat Biotechnol 2008, 26:771–774. Received: 19 December 2012 Accepted: 30 April 2013 27. Endy D: Foundations for engineering biology. Nature 2005, 438:449–453. Published: 10 May 2013 28. Guo L, Liang T, Lu Z: A comprehensive study of multiple mapping and feature selection for correction strategy in the analysis of small RNAs References from SOLiD sequencing. Biosystems 2011, 104:87–93. 1. Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, et al: Production of 29. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, et al: Breaking the code of the antimalarial drug precursor artemisinic acid in engineered yeast. DNA binding specificity of TAL-type III effectors. Science 2009, 326:1509–1512. Nature 2006, 440:940–943. 30. Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson 2. Ajikumar PK, Xiao WH, Tyo KE, Wang Y, Simeon F, et al: Isoprenoid pathway H, et al: Complete chemical synthesis, assembly, and cloning of a optimization for Taxol precursor overproduction in Escherichia coli. Mycoplasma genitalium genome. Science 2008, 319:1215–1220. Science 2010, 330:70–74. 31. Jaschke PR, Lieberman EK, Rodriguez J, Sierra A, Endy D: A fully 3. Colin VL, Rodriguez A, Cristobal HA: The role of synthetic biology in the decompressed synthetic bacteriophage oX174 genome assembled and design of microbial cell factories for biofuel production. J Biomed archived in yeast. Virology 2012, 434:278–284. Biotechnol 2011, 2011:601834. 32. Rai A, Boyle J: Synthetic biology: caught between property rights, the 4. Georgianna DR, Mayfield SP: Exploiting diversity and synthetic biology for public domain, and the commons. PLoS Biol 2007, 5:e58. the production of algal biofuels. Nature 2012, 488:329–335. 33. Calvert J: Ownership and sharing in synthetic biology: a ‘diverse ecology’ 5. Bonnet J, Subsoontorn P, Endy D: Rewritable digital data storage in live of the open and the proprietary. BioSocieties 2012, 7:169–187. cells via engineered control of recombination directionality. Proc Natl 34. Oye KA, Wellhausen R: The intellectual commons and property in Acad Sci U S A 2012, 109:8884–8889. synthetic biology.InSynthetic Biology: The Technoscience and Its Societal 6. Church GM, Gao Y, Kosuri S: Next-generation digital information storage Consequences. Edited by Schmidt M, Kelle A, Ganguli-Mitra A, de Vriend H. in DNA. Science 2012, 337:1628. Heidelberg, Germany: Springer; 2009:121–140. Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 17 of 18 http://www.jbioleng.org/content/7/1/13

35. Rutz B: Synthetic biology and patents. A European perspective. EMBO Rep 61. Knight T: Idempotent Vector Design for Standard Assembly of Biobricks. MIT 2009, 10(Suppl 1):S14–S17. Synthetic Biology Working Group Technical Report. 2003. http://dspace.mit. 36. Henkel J, Maurer SM: Parts, property and sharing. Nat Biotechnol 2009, edu/handle/1721.1/21168. 27:1095–1098. 62. Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, et al: BglBricks: A 37. Bertani G: Studies on lysogenesis. I. The mode of phage liberation by flexible standard for biological part assembly. J Biol Eng 2010, 4:1. lysogenic Escherichia coli. J Bacteriol 1951, 62:293–300. 63. Phillips IE, Silver PA: BBF RFC 23: A New BioBrick Assembly Strategy Designed for 38. Hollander DH, Nell EE: Improved preservation of Treponema pallidum and Facile Protein Engineering. 2006. http://dspace.mit.edu/handle/1721.1/32535. other bacteria by freezing with glycerol. Appl Microbiol 1954, 2:164–170. 64. Muller KM, Arndt KM, iGEM 2007 Team Freiburg, Grunberg R: BBF RFC 25: 39. Howard DH: The preservation of bacteria by freezing in glycerol broth. Fusion Protein (Freiburg) BioBrick assembly standard. 2009. http://dspace.mit. J Bacteriol 1956, 71:625. edu/handle/1721.1/45140. 40. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, et al: 65. Peisajovich SG, Horwitz A, Hoeller O, Rhau B, Lim WA: BBF RFC 28: A method Enzymatic assembly of DNA molecules up to several hundred kilobases. for combinatorial multi-part assembly based on the Type IIs restriction enzyme Nat Methods 2009, 6:343–345. AarI. 2009. http://dspace.mit.edu/handle/1721.1/46721. 41. Synthetic Genomics, Inc. Press Release: February 7, 2012. Synthetic Genomics, 66. Quan J, Tian J: Circular polymerase extension cloning of complex gene Inc. Announces Agreement with New England Biolabs to Launch Gibson libraries and pathways. PLoS One 2009, 4:e6441. Assembly™ Master Mix Product for Synthetic and Molecular Biology 67. Shao Z, Zhao H, Zhao H: DNA assembler, an in vivo genetic method for Applications. Available: http://www.syntheticgenomics.com/media/press/ rapid construction of biochemical pathways. Nucleic Acids Res 2009, 37:e16. 020712.html. Accessed 11.20.2012. 68. Hartley JL, Temple GF, Brasch MA: DNA cloning using in vitro site-specific 42. New England BioLabs, Inc., Gibson Assembly™ Master Mix, Limited Use Label recombination. Genome Res 2000, 10:1788–1795. License. Available: http://www.neb.com/nebecomm/products_intl/ 69. Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P, Fernandez-del productE2611.asp. Accessed 11.20.2012. -Carmen A, et al: GoldenBraid: an iterative cloning system for standardized 43. ATCC Press Release: June 18, 2012. ATCC Announces Agreement with Life assembly of reusable genetic modules. PLoS One 2011, 6:e21622. Technologies Corporation to Sell and Distribute Green Fluorescent Protein 70. Engler C, Kandzia R, Marillonnet S: A one pot, one step, precision cloning (GFP)-based Materials. Available: http://www.prweb.com/releases/2012/6/ method with high throughput capability. PLoS One 2008, 3:e3647. prweb9612892.htm Accessed 11.29.2012. 71. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S: A modular cloning 44. Mutalik VK: Expression Operating Unit: Origins and Current Status, BIOFAB system for standardized assembly of multigene constructs. PLoS One Community Meeting. 2010. June 19, 2010. http://www.biofab.org/sites/ 2011, 6:e16765. default/files/BIOFAB_CommunityMeeting1_Vivek.pdf. 72. Zhang Y, Werling U, Edelmann W: SLiCE: a novel bacterial cell extract- 45. Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, et al: based DNA cloning method. Nucleic Acids Res 2012, 40:e55. Precise and reliable gene expression via standard transcription and 73. Li MZ, Elledge SJ: Harnessing homologous recombination in vitro to translation initiation elements. Nat Methods 2013, 10:354–360. generate recombinant DNA via SLIC. Nat Methods 2007, 4:251–256. 46. Mutalik VK, Guimaraes JC, Cambray G, Mai QA, Christoffersen MJ, et al: 74. Carr PA, Church GM: Genome engineering. Nat Biotechnol 2009, 27:1151–1162. Quantitative estimation of activity and quality for collections of 75. Klock HE, Lesley SA: The polymerase incomplete primer extension (PIPE) functional genetic elements. Nat Methods 2013, 10:347–353. method applied to high-throughput cloning and site-directed 47. Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, et al: Measuring mutagenesis. Methods Mol Biol 2009, 498:91–103. the activity of BioBrick promoters using an in vivo reference standard. 76. Bitinaite J, Rubino M, Varma KH, Schildkraut I, Vaisvila R, et al: USER friendly J Biol Eng 2009, 3:4. DNA engineering and cloning method by uracil excision. Nucleic Acids 48. Rai A: Unstandard standarization: the case of biology. Communications of Res 2007, 35:1992–2002. the ACM 2010, 53:37–39. 77. Endy D, Deese I, MIT Synthetic Biology Working Group, and illustrated by 49. Torrance AW, Kahl LJ: Bringing standards to life: synthetic biology standards Wadey C: Adventures in Synthetic Biology. A comic book. 2005. available and intellectual property. Santa Clara Computer & High Tech L J. 2013. in press. online at http://dspace-test.mit.edu/handle/1721.1/46337. 50. Kumar S, Rai A: Synthetic biology: the intellectual property puzzle. 78. Velten L, Iwamoto N, Hiller C, Uckelmann H, Zhu C, et al: BBF RFC 41: Units Texas Law Review 2007, 85:1745–1768. for Promoter Measurement in Mammalian Cells. 2009. http://dspace.mit.edu/ 51. Bekkers R, West J: The limits to IPR standardization policies as evidenced handle/1721.1/49501. by strategic patenting in UTMS. Telecommunications Policy 2009, 33:80–97. 79. Galdzicki M, Wilson ML, Rodriguez CA, Pocock MR, Oberortner E, et al: BBF 52. Bekkers R, Iverson E, Blind K: Emerging ways to address the reemerging RFC 87: Synthetic Biology Open Language (SBOL) Version 1.1.0. 2012. http:// conflict between patenting and technological standardization. Indus Corp dspace.mit.edu/handle/1721.1/73909. Change 2012, 21:901–931. 80. JohnsonJ,GaldzickiM,SauroHM:BBF RFC 68: Standard for the Electronic 53. U.S. Dep’t of Justice, Fed Trade Comm'n: Antitrust Enforcement and Distribution of SBOLv Diagrams. 2010. http://dspace.mit.edu/handle/1721.1/60086. Intellectual Property Rights: Promoting Innovation and Competition. 2007. 81. Quinn JP, Beal J, Bhatia S, Cai P, Chen J, et al: BBF RFC 93: Synthetic Biology http://www.usdoj.gov/atr/public/hearings/ip/222655.pdf. Open Language Visual (SBOL Visual), version 1.0.0. 2013. http://dspace.mit. 54. U.S. Dep’t of Justice, U.S. Patent and Trademark Office: Policy Statement on edu/handle/1721.1/78249. Remedies for Standards-Essential Patents Subject to Voluntary F/Rand 82. Canton B, Labno A, Endy D: Refinement and standardization of synthetic Commitments. 2013. http://www.justice.gov/atr/public/guidelines/290994.pdf. biological parts and devices. Nat Biotechnol 2008, 26:787–793. 55. Oldham P, Hall S, Burton G: Synthetic biology: mapping the scientific 83. Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, et al: BglBrick vectors landscape. PLoS One 2012, 7:e34368. and datasheets: a synthetic biology platform for gene expression. J Biol 56. Carlson R: The changing economics of DNA synthesis. Nat Biotechnol Eng 2011, 5:12. 2009, 27:1091–1094. 84. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al: Gapped 57. U.S. Department of Health and Human Services, National Institutes of BLAST and PSI-BLAST: a new generation of protein database search Health: NIH Guidelines for Research Involving Recombinant DNA Molecules, programs. Nucleic Acids Res 1997, 25:3389–3402. revised October 2011. available at http://oba.od.nih.gov/rdna/ 85. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al: nih_guidelines_oba.html. Clustal W and clustal X version 2.0. Bioinformatics 2007, 23:2947–2948. 58. U.S. Department of Health and Human Services, National Institutes of 86. Kibbe WA: OligoCalc: an online oligonucleotide properties calculator. Health: Screening Framework Guidance for Synthetic Double-Stranded DNA Nucleic Acids Res 2007, 35:W43–W46. Providers, October 2010. available at http://www.phe.gov/Preparedness/legal/ 87. Stranneheim H, Lundeberg J: Stepping stones in DNA sequencing. guidance/syndna/Documents/syndna-guidance.pdf. Biotechnol J 2012, 7:1063–1073. 59. Smolke CD: Building outside of the box: iGEM and the BioBricks 88. Bertani G: Lysogeny at mid-twentieth century: P1, P2, and other foundation. Nat Biotechnol 2009, 27:1099–1102. experimental systems. J Bacteriol 2004, 186:595–600. 60. Carlson JM, Heckerman D, Shani G: Estimating false discovery rates for 89. Kocagoz T, Altin S, Turkyilmaz O, Tas I, Karaduman P, et al: Efficiency of the contingency tables. Microsoft Research Tech Report MSR-TR-2009-53. 2009. TK culture system in the diagnosis of tuberculosis. Diagn Microbiol Infect available at http://research.microsoft.com/apps/pubs/?id=80571. Dis 2012, 72:350–357. Kahl and Endy Journal of Biological Engineering 2013, 7:13 Page 18 of 18 http://www.jbioleng.org/content/7/1/13

90. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science 1994, 263:802–805. 91. Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, et al: Programming cells by multiplex genome engineering and accelerated evolution. Nature 2009, 460:894–898. 92. Slusarczyk AL, Lin A, Weiss R: Foundations for the design and implementation of synthetic genetic circuits. Nat Rev Genet 2012, 13:406–420.

doi:10.1186/1754-1611-7-13 Cite this article as: Kahl and Endy: A survey of enabling technologies in synthetic biology. Journal of Biological Engineering 2013 7:13.

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